CN113197894B - Application of olbatik in preparation of broad-spectrum anti-coronavirus medicines - Google Patents

Abstract

The invention discloses the preparation of Olbatik (OLX)Application in preparing broad-spectrum anti-coronavirus medicine. The invention finds that OLX has high-efficiency inhibition effect on MHV-A59 under nanomolar concentration, and meanwhile, the cytotoxicity of OLX is very low. It was further found that OLX acts at the stage of entry of coronavirus into cells, and prevents the entry of virus into cells, and is a virus entry inhibitor. In addition, when OLX is used at a concentration of 0.5. mu.M, the inhibitory effect on SARS-like coronavirus SARS-like CoV WIV1 and recombinant MERS-CoV live virus at the cellular level is about 95%. At the same time, OLX can inhibit the replication, EC, of 2019-nCoV WIV04 virus ₅₀ 398nM shows that olbatik has broad-spectrum coronavirus preventing or treating activity, important development value and wide application value.

Description

Application of olbatik in preparation of broad-spectrum anti-coronavirus medicines

Technical Field

The invention belongs to the technical field of medicines, in particular to a new application of Obatrox (Obatoclax Mesylate) in preparing broad-spectrum anti-coronavirus medicines, and in particular relates to a new application of Obatrox in preparing anti-human coronavirus, such as SARS-CoV, MERS-CoV, 2019-nCoV, HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV-HKU1 and the like; other vertebrate coronavirus, such as transmissible gastroenteritis virus (TGEV), epidemic diarrhea virus (PEDV), acute diarrhea syndrome coronavirus (SADS-CoV), and Infectious Bronchitis Virus (IBV). The invention encompasses the use of olbatik, alone or in combination, in the prophylaxis or treatment of coronaviruses.

Background

Since this century, several outbreaks and epidemics of highly pathogenic human coronaviruses have occurred intermittently, including the SARS coronavirus (SARS-CoV), the mors coronavirus (MERS-CoV) and the novel coronavirus (2019-nCoV). These pathogens infect humans and cause acute respiratory diseases. Besides the above-mentioned high pathogenic coronavirus, other human coronavirus such as HCoV-NL63, HCoV-229E, HCoV-OC43, HCoV-HKU1, etc. can cause slight upper respiratory diseases when infected by human, and are low pathogenic viruses, and people with low immunity are susceptible. In addition, coronavirus has a serious threat to livestock and poultry breeding, and typical viruses include transmissible gastroenteritis virus (TGEV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), Porcine Epidemic Diarrhea Virus (PEDV), Infectious Bronchitis Virus (IBV), and the like. It can be seen that coronavirus is an important pathogen with a wide host range and a very high risk.

Since outbreaks of highly pathogenic coronaviruses often cause huge social panic in a short time, seriously threaten human health and life safety, and have the risk of new and recurrent outbreaks, coronaviruses are widely concerned by researchers in global science, and the evolution process, pathogenic structure, pathogenic mechanism and the like of the coronaviruses are increasingly clarified. However, no specific drug against coronavirus infection is currently available. In the clinical treatment of SARS and MERS, the antiviral drugs mainly used include ribavirin, lopinavir, ritonavir, glucocorticoid, interferon or the combination of these drugs, but the treatment effect is limited or no effect at all, and these drugs have the risk of sequelae. Therefore, the development of highly effective specific anti-coronavirus drugs is a major and arduous task.

Obatoclax Mesylate, the Chinese translation name olbatox Mesylate or olbatox. It is also known as GX15-070, hereinafter referred to as OLX, by Gemin X. OLX is an antagonist of Bcl-2 family protein, can inhibit anti-apoptosis pathway mediated by Mcl-1 protein, and has effect of inducing cancer cell apoptosis. OLX has been validated for safety and therapeutic efficacy as an antitumor drug in first and second phase clinical studies in the united states. In recent years, it has been found that OLX has a specific antiviral function, and has been proved to have inhibitory effects on influenza a virus, several viruses of the alphavirus genus (including chikungunya virus and sindbis virus) and zika virus, but there has been little research on whether OLX has inhibitory effects on viruses of other genera.

Disclosure of Invention

In order to overcome the defects in the prior art, the invention aims to provide a new application of olbuterol mesylate, namely an application of olbuterol mesylate in preparing broad-spectrum anti-coronavirus medicines. The invention screens a high-flux compound library through an MHV-A59 coronavirus infection system to screen a drug Obatoclax Mesylate (also known as GX15-070, hereinafter referred to as OLX) approved by the United states Food and Drug Administration (FDA). OLX was found to have a high inhibitory effect on MHV-A59 at nanomolar concentrations, while OLX was found to be very low cytotoxic. It was further found that OLX acts at the stage of entry of coronavirus into cells, and prevents the entry of virus into cells, and is a virus entry inhibitor. In addition, OLX has inhibitory effect on SARS-like CoV WIV1, MERS-CoV, 2019-nCoV and other highly pathogenic coronaviruses, which indicates that OLX is a broad-spectrum medicine for preventing or treating coronaviruses.

The second purpose of the invention is to provide the application of the pharmaceutical composition containing olbatik mesylate in preparing the drugs for preventing or treating coronavirus.

In order to achieve the purpose, the invention provides the following technical scheme:

in a first aspect, the use of olbatik mesylate or a derivative thereof shown in formula (I) for preparing a medicament for preventing or treating coronavirus is provided,

preferably, in the above application, the olbatik mesylate derivative comprises a pharmaceutically acceptable salt thereof, or a compound with equivalent functions formed by reducing or increasing one or more groups in the structure of the formula (1).

In a second aspect, there is provided the use of a pharmaceutical composition comprising olbatik mesylate or a derivative thereof, as shown in formula (I), in the manufacture of a medicament for the prophylaxis or treatment of coronavirus.

Preferably, in the above application, the composition further comprises other antiviral drugs.

Preferably, in the above application, the composition further comprises a pharmaceutically acceptable carrier or excipient.

Preferably, in the above application, the coronavirus is a Human-infecting coronavirus selected from Severe acute respiratory syndrome coronavirus SARS-CoV (Severe acute respiratory syndrome coronavirus, SARS-CoV), Middle East respiratory syndrome coronavirus MERS-CoV (Middle East respiratory syndrome coronavirus or syndrome coronavirus, MERS-CoV), novel coronavirus 2019-nCoV, Human coronavirus HCoV-NL63(Human coronavirus NL63), Human coronavirus HCoV-229E (Human coronavirus 229E), Human coronavirus HCoV-OC43(Human coronavirus OC43), Human coronavirus HCoV-HKU1(Human coronavirus HKU 1); other vertebrate coronaviruses are selected from porcine infectious gastroenteritis virus (TGEV), Porcine Epidemic Diarrhea Virus (PEDV), porcine acute diarrhea syndrome coronavirus (SADS-CoV), Infectious Bronchitis Virus (IBV), and mouse hepatitis virus MHV-A59.

Further preferably, in the above application, the coronavirus is SARS-CoV, MERS-CoV, novel coronavirus 2019-n CoV, mouse hepatitis virus MHV-A59.

In a third aspect, there is provided the use of olbuterol mesylate shown in formula (I) or a derivative thereof or a pharmaceutical composition containing olbuterol mesylate shown in formula (I) or a derivative thereof in the preparation of a medicament for inhibiting the entry of a coronavirus into a host cell.

The compounds of the present invention or pharmaceutical compositions containing them may be administered in unit dosage form by enteral or parenteral routes, such as oral, intravenous, intramuscular, subcutaneous, nasal, oromucosal, ophthalmic, pulmonary and respiratory, dermal, vaginal, rectal, and the like.

The dosage form for administration may be a liquid dosage form, a solid dosage form, or a semi-solid dosage form. The liquid dosage forms can be solution (including true solution and colloidal solution), emulsion (including o/w type, w/o type and multiple emulsion), suspension, injection (including water injection, powder injection and infusion), eye drop, nose drop, lotion, liniment, etc.; the solid dosage form can be tablet (including common tablet, enteric coated tablet, buccal tablet, dispersible tablet, chewable tablet, effervescent tablet, orally disintegrating tablet), capsule (including hard capsule, soft capsule, and enteric coated capsule), granule, powder, pellet, dripping pill, suppository, pellicle, patch, aerosol (powder), spray, etc.; semisolid dosage forms can be ointments, gels, pastes, and the like.

The compound can be prepared into common preparations, sustained release preparations, controlled release preparations, targeting preparations and various particle drug delivery systems.

For tableting the compounds of the invention, a wide variety of excipients known in the art may be used, including diluents, binders, wetting agents, disintegrants, lubricants, glidants. The diluent can be starch, dextrin, sucrose, glucose, lactose, mannitol, sorbitol, xylitol, microcrystalline cellulose, calcium sulfate, calcium hydrogen phosphate, calcium carbonate, etc.; the wetting agent can be water, ethanol, isopropanol, etc.; the adhesive can be starch slurry, dextrin, syrup, Mel, glucose solution, microcrystalline cellulose, acacia slurry, gelatin slurry, carboxymethyl cellulose sodium, methyl cellulose, hydroxypropyl methyl cellulose, ethyl cellulose, acrylic resin, carbomer, polyvinylpyrrolidone, polyethylene glycol, etc.; the disintegrant can be dry starch, microcrystalline cellulose, low-substituted hydroxypropyl cellulose, crosslinked polyvinylpyrrolidone, crosslinked sodium carboxymethylcellulose, sodium carboxymethyl starch, sodium bicarbonate and citric acid, polyoxyethylene sorbitol fatty acid ester, sodium dodecyl sulfate, etc.; the lubricant and glidant may be talc, silicon dioxide, stearate, tartaric acid, liquid paraffin, polyethylene glycol, and the like.

The tablets may be further formulated into coated tablets, such as sugar-coated tablets, film-coated tablets, enteric-coated tablets, or double-layer and multi-layer tablets.

To encapsulate the administration units, the active ingredient of the compounds of the invention can be mixed with diluents and glidants and the mixture can be placed directly into hard or soft capsules. Or preparing the effective component of the compound of the invention into granules or pellets with a diluent, an adhesive and a disintegrating agent, and then placing the granules or pellets into hard capsules or soft capsules. The various diluents, binders, wetting agents, disintegrants, glidants used to prepare tablets of the compounds of the present invention may also be used to prepare capsules of the compounds of the present invention.

For preparing the compound of the present invention into injection, water, ethanol, isopropanol, propylene glycol or their mixture can be used as solvent, and appropriate amount of solubilizer, cosolvent, pH regulator, and osmotic pressure regulator commonly used in the art can be added. The solubilizer or cosolvent can be poloxamer, lecithin, hydroxypropyl-beta-cyclodextrin, etc.; the pH regulator can be phosphate, acetate, hydrochloric acid, sodium hydroxide, etc.; the osmotic pressure regulator can be sodium chloride, mannitol, glucose, phosphate, acetate, etc. For example, mannitol and glucose can be added as propping agent for preparing lyophilized powder for injection.

In addition, colorants, preservatives, flavors, or other additives may also be added to the pharmaceutical preparation, if desired.

For the purpose of administration, enhancing the therapeutic effect, the drug or pharmaceutical composition of the present invention can be administered by any known administration method.

The compounds or compositions of the present invention may be administered alone or in combination with other therapeutic or symptomatic agents. When the compound of the present invention acts synergistically with other therapeutic agents, its dosage should be adjusted according to the actual circumstances.

Has the advantages that:

in the present invention, when the concentration of OLX used is 0.005. mu.M-0.5. mu.M, it is effective in inhibiting MHV-A59(GenBank Accession: AY700211), [ solution of murine hepatitis virus ]1]Replication in host cells (Neuro-2a), EC ₅₀ 11.4nM, with very low cytotoxicity. Addition of the O LX drug 4-12 hours after MHV-A59 infection was also effective in reducing viral replication in the host cells (FIG. 2).

In the present invention, SARS-like CoV WIV1(GenBank Accession: KF367457.1) is administered to a bat-derived S ARS-like coronavirus at a cellular level at a concentration of 0.5. mu.M2]The inhibiting efficiency reaches 95 percent, and the recombinant MERS-CoV (GenBank Accession: MH734115), [ 2 ]3]The inhibition of live virus reaches 94%. Meanwhile, OLX can inhibit 2019-nCoV WIV04(GISAID AC accession No. EPI _ ISL _402124, national viral resources Bank, accession No. IVCAS 6.7512), (SEQ ID NO: IVCAS 6.7512)4]Replication of the virus, EC ₅₀ 398nM (FIGS. 4-6).

In the invention, a mouse acute hepatitis model is constructed by injecting mouse hepatitis virus MHV-A59 by liver, and is used for evaluating the anti-coronavirus effect of OLX at an animal level. This part of the results shows that, compared with the control mice (which only injected the virus and did not inject the drug), the two-time tail vein injection of 0.05 μ g/g OLX can effectively reduce the replication of MHV-A59 in the liver of the mice, relieve the symptoms of the mice caused by virus infection, and protect the mice from being killed by MHV-A59 infection (FIG. 7).

The safety of the medicine to human bodies is verified in first-stage and second-stage clinical researches in the United states, the obvious effect of olbatik mesylate on broad-spectrum anti-coronavirus is determined, the long period of research and development of a new medicine is avoided, powerful theoretical basis and practical basis are provided for further research and development of anti-coronavirus, particularly anti-novel coronavirus 2019-nCoV, and the medicine has important development value and popularization significance.

The compound Obatoclax Mesylate used in the present invention is recognized as Obatrox Mesylate, or Obatrox. It is also known as GX15-070, which is found by Gemin X. Olbatox mesylate was purchased from shelck, cat # S1057. The web page links intohttps://www.selleck.cn/products/Obatoclax-Mesylate.html。

Drawings

FIG. 1 shows the effect of 4 small-molecule drugs on MHV-A59 replication and its cytotoxicity. The results show that the drug OLX has high antiviral effect and low cytotoxicity.

A: the effect of 4 kinds of small molecule drugs (W-77, W-24, W-13, OLX) obtained by primary screening on MHV-A59 virus replication. The solvent DMSO (concentration 0.1%) of 4 drugs was used as a control, and MHV-A59 virus was added simultaneously with the drug at the corresponding concentration. And (5) after the adsorption of the virus is finished, replacing the fresh culture medium, and simultaneously continuously adding the medicine with the corresponding concentration until the sample is collected. Cell supernatants were harvested 16h after virus infection and tested for virus titer using the plaque assay. The concentrations of the four drugs are W-77: 3 μ M, W-24: 3 μ M, W-13: 20 μ M, OLX: 0.5 mu M; MOI is 0.1 and cell line is Neuro-2 a. As can be seen from the results, drugs W-13 and OLX can significantly inhibit the replication of MHV-A59 in host cells at different concentrations, and the effect of OLX is most obvious.

B: the cytotoxicity of the W-13 and OLX drugs was examined. And adding a corresponding concentration of drug into Neuro-2a cells by taking 0.1% DMSO as a control, and detecting the activity of the cells by using a CCK8 kit after incubation for 48 hours. After adding CCK8 reagent and incubating for 1h, detecting the absorbance of cell fluid at 450nm, wherein the larger value represents the better activity of the cell. The results indicate that the drug W-13 is very cytotoxic in the concentration range (20. mu.M) where it exerts its antiviral effect. When the concentration of OLX reaches 2 muM, the cytotoxicity is still very low (CC50>2 muM), and the OLX in the range can play a high-efficiency antiviral role, which indicates that the OLX not only has a remarkable anti-coronavirus effect, but also has very high safety. We therefore chose OLX as the target for further studies.

FIG. 2 shows the inhibitory effect of OLX on the replication of MHV-A59 of coronavirus.

A: OLX anti-MHV-A59 virus EC ₅₀ And (4) measuring. And adding different concentrations of OLX while adding MHV-A59 virus by taking 0.1% DMSO as a control, changing a fresh culture medium after adsorption is finished, and continuously adding the same concentration of drug to incubate the cells until the cells are sampled. Supernatants were harvested 16h post infection and assayed for viral titer by plaque assay. MOI is 0.1 and cell line is Neuro-2 a. As can be seen from the figure, OLX inhibits the EC of MHV-A59 ₅₀ 11.4nM, very good anti-coronavirus effect at very low drug concentration and dose-dependent antiviral effect. Results FIG. 1B, its SI (CC50/EC50)>175。

B: inhibitory effect of OLX on MHV-A59 replication following viral infection. MHV-a59 virus solution was added to Neuro-2a cells without the addition of drug, MOI 0.1. And (4) after adsorption, discarding virus liquid, washing cells by PBS, and replacing fresh culture medium. OLX was added at a concentration of 0.1. mu.M at 4, 8 and 12 hours after virus infection, respectively, with the time of virus addition as the infection starting point. After 16 hours of drug incubation, supernatants were harvested and virus titers were detected by plaque assay. In addition, cells are lysed by adding Trizol, total RNA of the cells is extracted, and the relative expression quantity of MHV-A59 mRNA7 is detected by a q-PCR method after reverse transcription. At the mRNA level, the mRNA7 change was not significant after 4h of viral infection with OLX, but the antiviral effect of OLX was very significant after 8h of infection.

C: inhibitory effect of OLX on MHV-A59 replication following viral infection. After the above treatment, the supernatant was collected and the virus titer was measured by plaque assay. The results show that at the level of live virus, OLX can be added at 4, 8 and 12h after infection to show very good antiviral effect. The above results indicate that the drug OLX can still function after viral infection.

FIG. 3 is a preliminary study of OLX anti-coronavirus effect nodes, and analysis of drug effect time nodes shows that OLX plays a role in the virus entry stage.

The MOI was taken to be 0.1, OLX was added at a concentration of 0.1 μ M at different time nodes before and after MHV-a59 virus infection, and the cells were incubated for different time periods and the cell supernatants were collected after 8 hours of virus infection. The samples were then tested for viral titer by plaque assay. A represents a time node diagram. B shows the inhibitory effect of OLX after incubation at various times before and after viral infection. The results show that, when the drug is added 2 hours before infection, the inhibitory effect of OLX (-2to 0h) existing during the virus adsorption entry process is 2.5 times that of the inhibitory effect of OLX (-2to attach) not existing during the adsorption entry process. In addition, when the drug is administered after infection, the effect of the drug administration is the worst in 0-1h (0to 1) and 4-8h (4to 8), and the inhibitory effect of the drug administration is similar in 0-8h (0to 8) and 2-8h (2to 8). MHV has a time between 2 and 4 hours to complete one round of replication and release in Neuro-2a cells, so that the presence of OLX for 0to 8 hours or 2to 8 hours can just inhibit the second round of infection of the virus released outside the cells, which can explain why the addition of OLX at the above time has a better inhibitory effect. And in 0-1h or 4-8h, most of viruses in the first round or the second round are completely entered, and the medicine cannot play a role in inhibiting the entry of the viruses. Thus, OLX acts at the entry stage of the virus and the anti-coronavirus effect of OLX is associated with the inhibition of viral entry.

FIG. 4 shows the inhibitory effect of OLX on SARS-like coronavirus WIV 1. SARS-like CoV WIV1 is a coronavirus isolated from bat, and is very similar to SARS-CoV, and uses ACE2 as a receptor, and has the ability to infect human.

OLX was added at a concentration of 0.5mM while adsorbing SARS-like CoV WIV 1. After adsorbing for 2h, the virus solution was discarded, the cells were washed with PBS, the fresh medium was replaced, and OLX of the same concentration was added continuously for incubation. Cell supernatants were collected 24 hours after the adsorption was completed and the viral titer of SARS-like CoV WIV1 was measured by plaque assay. From the results, 0.5 μ M OLX can effectively inhibit the replication of SARS-like CoV WIV1, and the inhibition effect can reach 95%.

FIG. 5 shows the inhibitory effect of OLX on MERS-CoV.

MERS-CoV used in this experiment is a recombinant live virus [ alpha ], [ alpha ] or3]. MERS-CoV adsorption was added along with OLX at a concentration of 0.5. mu.M. After adsorbing for 2h, the virus solution was discarded, the cells were washed with PBS, the fresh medium was replaced, and OLX of the same concentration was added continuously for incubation. And (5) respectively collecting cell supernatant and cell repeated freezing and thawing solution 24 hours after adsorption is finished, and detecting the MERS-CoV virus titer by a plaque method. The figure shows the plaque experiment detection result. From the results, 0.5. mu.M OLX inhibited MERS-CoV replication at 94% efficiency.

FIG. 6 shows the inhibitory effect of OLX on 2019-nCoV WIV04 and EC ₅₀ And (4) measuring.

2019-nCoV WIV04 is a strain isolated from a sample of ICU patients (national viral resources Bank, accession number: IVCAS 6.7512) [4]]. The 2019-nCoV WIV04 virus infects Vero E6 cells with the MOI of 0.01, adds OLX with different concentrations while adding the virus, changes the fresh culture medium after the adsorption is finished, and simultaneously continues adding the same concentration of drug to incubate the cells until the cells are collected. 0.1% DMSO was used as a control in the experiment. After 24h, cell RNA is extracted, and the virus replication level is detected by using an RT-qPCR method. OLX inhibits EC of 2019-nCoV WIV04 ₅₀ 398 nM.

FIG. 7 shows the protective effect of OLX on the level of MHV infected C57BL/6 mice. C57BL/6 mice were injected into the liver 10 ⁶ PFU MHV, and the intraperitoneal injection method is used for injecting corresponding dose on the 0 th day and the 2 nd day. The dose per group was 0.01. mu.g/g and 0.05. mu.g/g, respectively.

A: the body weight of the mice changed. After administration, half of the mice (n-5) were observed for survival and recorded, along with changes in body weight. As can be seen from the figure, the weight loss values of the two groups of mice injected with the drug were smaller than those of the mice of the drug-free control group.

B: survival rate of mice. From the survival rate of mice in each group, the overall survival rate of mice in the 0.01. mu.g/g OLX group was higher than that of mice in the OLX-free group, but lower than that of mice in the 0.05. mu.g/g OLX group. Mice in the 0.05. mu.g/g OLX group were all alive and this dose completely protected mice from death due to MHV infection.

C: mouse liver virus titer. The other half of the mice (n ═ 5) were dissected 3 days after infection, livers were removed, weighed, ground to homogenate and centrifuged, and supernatants were removed to test for viral titer in the tissues. The results show that the doses of 0.01 mu g/g and 0.05 mu g/g can effectively reduce the MHV replication in the liver of a mouse, and the virus inhibition effect is better along with the increase of the doses.

Detailed Description

The features and advantages of the present invention will be further understood from the following detailed description taken in conjunction with the accompanying drawings. The examples provided are merely illustrative of the method of the present invention and do not limit the remainder of the disclosure in any way.

The following are routine experiments referred to in the examples:

1. cell culture

The cell lines used in this experiment were Neuro-2a and L2, provided by the laboratory. The cells were cultured in DMEM medium containing 10% FBS and 1% penicillin and streptomycin at 37 deg.C in an incubator under CO atmosphere ₂ The concentration was 5%.

2. Viral amplification

The MHV virus required for this experiment needs to be amplified in Neuro-2a cells. Inoculating Neuro-2a cells into a 10cm dish, adding virus mother liquor after the cells grow to 70-90% of density, adsorbing for 2h, and replacing with fresh culture medium. Observing the degree of cytopathic effect, and harvesting the virus by adopting a repeated freeze-thaw method after 75 percent of syncytia formed by virus infection (generally 16-24 hours). Cell debris was removed by centrifugation at 1000rpm, and the virus was stored frozen at-80 ℃ after packaging.

3. Plaque assay for viral titer

Plaque assay (plaque assay), also known as plaque assay, is a common method used to determine virus titer in virology experiments. The principle is simple, the result is reliable, and the amount of live virus particles which can infect cells to achieve proliferation is measured. If the culture environment of the cells is a liquid environment, the virus released into the environment can freely flow to various sites to infect other cells. If the culture environment of the cells is changed into a solid or semi-solid culture medium, the virus released into the environment cannot spread freely and only stays at the original position to infect other cells nearby the original host. Thus, after several rounds of replication, the virus will infect all cells in the vicinity of the originally infected host and then die, forming a "plaque" without cells. When staining is performed with a staining reagent (e.g., crystal violet or neutral red), the cells are stained and the plaques are not stained, so that the total number of viruses originally infecting these cells can be counted. The calculated result is called Plaque Forming Unit (PFU), and how many plaques can be formed per unit can be regarded as the number of live viruses.

In practical operation, firstly, the virus solution to be tested needs to be diluted by 10 times continuously, and then the diluted solution is added into monolayer cells which can form plaques in sequence for adsorption for 1-2 h. After adsorption, covering the cells with a solid or semi-solid culture medium, culturing in an incubator at 37 ℃, staining after forming macroscopic plaques, then recording the number of the plaques, and calculating the number of viruses in the stock solution according to the dilution factor and the sample loading volume. In this experiment, the titer of MHV was measured using L2 cells. Virus liberation was limited using 1.9% agar medium and cells were stained with neutral red solution to calculate plaques. The concrete steps are as follows (taking MHV-A59 as an example):

a. cell plating: l2 cells were seeded into six well plates, six wells prepared for one sample. The cells are cultured to reach a density of more than 90%, and the culture time is not more than two days so as to avoid cell aging.

b. Diluting virus liquid: 6 1.5mL centrifuge tubes were prepared and 900. mu.L serum free Medium DMEM, numbered 1-6, was added to each centrifuge tube to determine the dilution titer of 10 ^-1 To 10 ^-6 . And (3) fully and uniformly mixing the virus liquid with the original concentration, sequentially absorbing 100 mu L of virus liquid for gradient dilution, fully shaking and uniformly mixing. Repeating the steps, and adding the uniformly mixed virus liquid into the centrifugal tube with the next serial number. Until the final drop is dilutedAnd (4) degree. Mixing well for use.

c. Virus adsorption: cells cultured to a density of 90% or more were removed, and the old medium was discarded. The diluted virus solution was added to a six-well plate. The cells are placed in an incubator for adsorption for 1-2 h.

d. Preparing a solid culture medium: heating to melt agar, and placing in 55 deg.C water bath to cool and equilibrate. A2 × DMEM medium was prepared, and 10% fetal bovine serum was added to prepare a serum-containing 2 × DMEM medium. Subpackaging the 2x DMEM medium into centrifuge tubes, and warming for later use.

e. Adding a solid culture medium: after the adsorption was completed, the supernatant was aspirated. Cells were rinsed once with either pre-warmed PBS or serum-free DMEM. Adding agar into preheated 2x DMEM medium, quickly mixing uniformly, and adding into a six-well plate.

f. Culturing: and (5) placing the culture medium into an incubator for continuous culture after the culture medium is solidified.

g. And (3) dyeing observation counting: after incubation until macroscopic plaques are formed, staining is performed. Serum-free DMEM is used as a solvent, 0.7% neutral red preservation solution is prepared into staining solution containing 6% neutral red preservation solution, and the staining solution is added into the holes. And after continuing culturing for 6-8h, digging out the solid culture medium, counting the plaques, and calculating the virus titer according to the counting result.

4. Real-time quantitative PCR detection of virus titer

In the present invention, we also measure the replication level of the virus by measuring the amount of the viral genome, in addition to the plaque method. Since the viral genome is RNA, which is difficult to measure, the viral genome is first reverse transcribed into cDNA and then measured.

1) Extracting RNA in cells:

a. an appropriate amount of TRIzol (1 mL in a 6-well plate) was added to the cells, and the cells were disrupted by repeatedly pipetting with a pipette tip. Standing the cell fluid frozen at-80 deg.C at room temperature for 5-10min, and recovering to room temperature.

b. 0.2 volume of chloroform (relative to the initial volume of TRIzol) was added to the lysate, and centrifuged vigorously for 15 sec at 12,000rpm and 4 ℃ for 15 min.

c. The upper aqueous phase (about 60% of the initial TRIzol volume) was transferred to a new EP tube, mixed with 0.5 volume of isopropanol and left at room temperature for 5-10min to precipitate RNA. After the precipitation was completed, the mixture was centrifuged at 12,000rpm at 4 ℃ for 10 min.

d. The supernatant was removed, an appropriate amount of 75% ethanol was added, and the RNA pellet was gently resuspended to remove protein impurities. Centrifuge at 12,000rpm at 4 ℃ for 10 min.

e. The supernatant was discarded and left at room temperature for several minutes until the RNA precipitate was dried. The RNA pellet was solubilized by adding an appropriate volume (50-100. mu.L) of RNase-free H2O and carefully pipetting to aid RNA solubilization if necessary.

f. A1. mu.L RNA sample was taken, and the RNA concentration was measured with a spectrophotometer and diluted to 100-300 ng/. mu.L for use in the reverse transcription reaction. The remaining RNA samples were frozen at-80 ℃.

2) Reverse transcription:

the kit used in this experiment was supplied by Invtrogen corporation. The following reverse transcription experimental procedure:

a. mu.g of RNA and 1. mu.g of oligo dT were mixed, and DECP water was added to 12. mu.L, followed by incubation at 65 ℃ for 5 min.

b. The sample was taken out and placed on ice for 2-3 min.

c. The following components were added to the sample:

after gentle mixing, the mixture was incubated at 42 ℃ for 1 hour.

3) Fluorescent quantitative PCR

a. Designing a primer: the primers involved in the invention are templates encoding MHV N proteins.

b. Fluorescent quantitative PCR system (20 μ L):

setting a PCR program:

95℃10min

95℃15sec

60℃30sec

repeat cycle 40 times starting from 2

Three identical parallel sets were set up for each sample.

5. Cytotoxicity test (CCK8 kit)

Cell Counting Kit-8 (CCK-8) is a reagent commonly used in laboratories for detecting and analyzing Cell proliferation and drug toxicity. It has simple operation and reliable result, and is widely applied to various cancers or medicines and other various related researches. The reagent contains WST-8, namely 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfonic acid benzene) -2H-tetrazole monosodium salt. This compound can be reduced by dehydrogenase in cells in the presence of an electron carrier 1-Methoxy PMS (i.e., 1-Methoxy-5-methylphenazinium dimethylsulfate) to form a yellow Formazan product (Formazan dye). The product is water soluble and yellow, and the amount of the product produced is proportional to the number of living cells. Thus, the reagent can be used to detect the number of living cells. The specific detection method comprises the following steps:

a. cell inoculation: cells were seeded into 96-well plates, requiring at least 3-4 replicate wells per drug concentration. The cells are placed in an incubator for culture, and experiments are carried out when the cells grow to about 80%.

b. And (3) incubation of the drugs: adding the corresponding medicine to be detected into each hole, and shaking and mixing uniformly after adding. The cells were then placed in an incubator for continued incubation. The incubation time is changed according to the experimental needs, and the toxicity of the detected drug is generally more than 24 hours.

And c, CCK8 reagent detection: the cells were removed, 10. mu.L of CCK8 reagent was added to each well, mixed well by shaking, and the cells were incubated in an incubator. After half an hour, the medium is taken out to see whether it has turned yellow. If the medium becomes yellow it can be detected immediately, otherwise incubation at 37 ℃ is continued until the cell culture medium becomes yellow. After the culture solution became yellow, absorbance at 450nm was measured. A larger value indicates better cell activity.

Example 1 detection of the Effect of the 4 small molecule drugs on MHV replication and the cytotoxicity (example MHV-A59)

1. Detecting the influence of the 4 kinds of small molecular drugs obtained by primary screening on MHV virus replication

Neuro-2a cells were plated in 12-well plates and experiments were performed up to 80% in length. This test was done in a BSL-2 laboratory environment. The virus was added at MOI ═ 0.1. Using 4 drugs (W-77, W-24, W-13, OLX) in DMSO (concentration 0.1%) as a control, MHV virus was added together with the drugs (W-77: 3. mu.M, W-24: 3. mu.M, W-13: 20. mu.M, OLX: 0.5. mu.M) at the corresponding concentrations. And (4) after the adsorption of the virus is finished, replacing the fresh culture medium, and simultaneously continuously adding the medicine with the corresponding concentration until the sample is collected. Cell supernatants were harvested 16h after virus infection and tested for virus titer using plaque assay. As can be seen from the results, drug W-13 was able to inhibit MHV replication in host cells at a concentration of 20. mu.M, while OLX was more effective in inhibiting MHV at a concentration of 0.5. mu.M (FIG. 1A).

2. Detection of cytotoxicity of drugs W-13 and OLX

Given that drugs W-13 and OLX have antiviral effects at different concentrations, we examined their cytotoxicity at the concentration ranges where they each exert an antiviral effect. And adding a corresponding concentration of drug into Neuro-2a cells by taking 0.1% DMSO as a control, and detecting the activity of the cells by using a CCK8 kit after incubation for 48 hours. After adding CCK8 reagent and incubating for 1h, detecting the absorbance of cell fluid at 450nm, wherein the larger value represents the better activity of the cell. As can be seen, the drug W-13 is very cytotoxic in the concentration range (20. mu.M) where it exerts its antiviral effect. However, when the concentration of OLX is below 0.5 μ M, cytotoxicity is not substantially detected, and OLX can exert a highly effective antiviral effect in this range, indicating that OLX not only has a significant anti-coronavirus effect, but also has very high safety (fig. 1B). We therefore chose OLX as the subject of the subsequent study.

Example 2 detection of the inhibition of MHV replication by OLX at various stages (example MHV-A59)

OLX against MHV VirusEC of (1) ₅₀ Measurement of

To define the effective concentration range of OLX for inhibiting the replication of MHV coronavirus, we determined the EC of OLX for inhibiting MHV virus ₅₀ The value is obtained. Neuro-2a cells were infected at MOI ═ 0.1. And (3) taking 0.1% DMSO as a control, adding MHV virus and gradient concentration OLX, changing a fresh culture medium after adsorption is finished, and continuously adding the same concentration of drug to incubate the cells until the cells are sampled. Supernatants were harvested 16h post infection and assayed for virus titer by plaque assay. The results show that OLX inhibits EC of MHV ₅₀ At 11.4nM, the effect was very good against coronavirus at a concentration of drug in nanomolar range, and the antiviral effect was dose-dependent (fig. 2A).

2. Detection of the inhibitory Effect of OLX on MHV replication after Virus infection

Since in the actual treatment of viral infections, it is often necessary to first confirm the viral infection and then use the drug symptomatically, we examined whether antiviral effects can be achieved by using OLX after MHV infection. MHV virus fluid was added to Neuro-2a cells at MOI ═ 0.1, at which time no drug was added. After adsorption, the virus solution was discarded, and the cells were washed with PBS and replaced with fresh medium. OLX was added at a concentration of 0.1. mu.M at 4, 8, and 12 hours after virus infection, respectively, with the time of virus addition as the infection starting point. After 16 hours of drug incubation, supernatants were harvested and virus titers were detected by plaque assay. Meanwhile, cells are lysed by adding Trizol, total RNA of the cells is extracted, and after being subjected to reverse transcription, the relative expression quantity of MHV mRNA7 is detected by a q-PCR method. The results show that at the level of mRNA7, the inhibitory effect of the addition of OLX was not significant 4h after viral infection, but after 8h infection, the antiviral effect of OLX was very significant and the MHV replication level was reduced by 1-fold compared to the control (FIG. 2B). At the live virus level, OLX was added 4, 8, 12h post-infection, and showed very good antiviral effect, inhibiting MHV replication by more than 3-fold compared to the control OLX (fig. 2C). The above results indicate that the drug OLX can still function after viral infection.

EXAMPLE 3 preliminary investigation of the mechanism of action of OLX against coronavirus (example MHV-A59)

To explore the specific role of OLX anti-coronavirus on which stage of virus replication, we performed a drug action time node analysis. Taking MOI (0.1) to infect Neuro-2a cells, adding OLX with the concentration of 0.1 mu M at different time points before and after MHV virus infection, and incubating for different time periods, wherein (-2to 0) is added with medicine two hours in advance, and is added with medicine when infecting virus; (-2to attach): adding medicine two hours in advance, removing medicine before infecting virus, and adding no medicine while infecting virus; (0to 1), adding the medicine again after the virus infection is finished, acting the medicine for 1 hour, and removing the medicine; (0to 8) adding the medicine after the virus infection is finished, and removing the medicine after the medicine acts for 8 hours; (2to 8) adding medicine after the virus infection is finished for 2 hours, and removing the medicine after the medicine acts for 6 hours; (4to 8) adding medicine after the virus infection is finished for 4 hours, acting the medicine for 4 hours, and removing the medicine. The cell supernatant was collected and then the virus titer in the sample was measured by plaque assay. The results show that, when the drug is added before infection, the inhibition effect of OLX (-2to 0h) existing in the adsorption process is 2.5 times that of OLX (-2to attach) not existing in the adsorption process. In addition, 0to 1h and 4to 8h were the least effective when dosed after infection, while 0to 8h and 2to 8h were similar in inhibitory effect (fig. 3). MHV completes one round of replication and is released in Neuro-2a cells for a period of time between 2 and 4 hours, so that the presence of OLX at 0to 8h or 2to 8h could just inhibit the second round of infection of the virus released outside the cells, which could explain why the addition of OLX at the above-mentioned time had a better inhibitory effect. Between 0to 1h or 4to 8h, the first round or the second round of virus mostly completes the entry, and the drug cannot play a role in inhibiting the entry. Thus, OLX acts at the entry stage of the virus and the anti-coronavirus effect of OLX is associated with inhibition of virus entry.

Example 4 detection of the inhibitory Effect of OLX on SARS-like coronavirus WIV1

SARS-like coronavirus is a coronavirus isolated from bat, has the ability to infect humans, and can cause mild respiratory disorders in humans, much like SARS-CoV. This test was done in a P3 laboratory environment. While adsorbing Vero cells by SARS-like coronavirus, OLX was added at a concentration of 0.5. mu.M. After adsorbing for 2h, the virus solution was discarded, the cells were washed with PBS, the fresh medium was replaced, and OLX of the same concentration was added continuously for incubation. And collecting cell supernatant 24 hours after adsorption is finished, and detecting the SARS-like virus titer by a plaque method. From the results, OLX was able to effectively inhibit the replication of SARS-Like coronavirus, and the inhibitory effect was up to 95% (FIG. 4).

Example 5 detection of inhibitory Effect of OLX on MERS-CoV coronavirus

MERS-CoV is a highly pathogenic coronavirus. This test was done in a P3 laboratory environment. MERS-CoV infected Caco2 cells at MOI ═ 0.1, and OLX was added at a concentration of 0.5 μ M while virus was adsorbed. After adsorbing for 2h, the virus solution was discarded, the cells were washed with PBS, the fresh medium was replaced, and OLX of the same concentration was added continuously for incubation. And (5) respectively collecting cell supernatant and cell repeated freezing and thawing solution 24 hours after adsorption is finished, and detecting the MERS-CoV virus titer by a plaque method. The figure shows the plaque experiment detection result. From the results, OLX was able to inhibit MERS-CoV replication, and the inhibitory effect reached 94% (FIG. 5).

Example 6 detection of the inhibitory Effect of OLX on 2019-nCoV WIV04 and its EC ₅₀ And (4) measuring.

2019-nCoV WIV04 is a strain isolated from a sample of ICU patients (national viral resources Bank, accession number: IVCAS 6.7512) [4 ]. This test was done in a BSL-3 laboratory environment. The 2019-nCoV WIV04 virus infects Vero E6 cells with MOI of 0.01, adds OLX with different concentration while adding the virus, changes the fresh culture medium after adsorption is finished, and simultaneously continues adding the same concentration of drug to incubate the cells until the cells are collected. With 0.1% DMSO as control. After the incubation is finished, extracting cell RNA, and detecting the virus replication level by using an RT-qPCR method. The targeted S gene was detected with the following primers (from reference 4):

RBD-qF1:5’-CAATGGTTTAACAGGCACAGG-3’；

RBD-qR1:5’-CTCAAGTGTCTGTGGATCACG-3。

the results show that OLX inhibits EC of 2019-nCoV WIV04 ₅₀ 398nM (FIG. 6).

Example 7 detection of the inhibitory Effect of OLX on MHV-A59 at the level of C57BL/6 mice and the protective Effect of OLX on MHV-infected C57BL/6 mice

This test was done in the ABSL II laboratory. SPF grade 3 week old male C57BL/6 mice were purchased and grouped according to the following experimental protocol:

intrahepatic injection 10 ⁶ PFU MHV was injected simultaneously with the tail vein injection at the day 0 and day 2 of infection (as above table), and the dose per group was 0.01. mu.g/g and 0.05. mu.g/g, respectively.

After administration, half of the mice (n-5) were observed for survival and recorded, along with changes in body weight. As can be seen from the figure, the weight loss values of the two groups of mice injected with the drug were smaller than those of the mice of the drug-free control group (fig. 7A). From the survival rate of mice per group, the overall survival rate of mice in the 0.01. mu.g/g OLX group was higher than that of mice in the OLX-free group, but lower than that of mice in the 0.05. mu.g/g OLX group. Mice in the 0.05. mu.g/g OLX group were all alive and this dose completely protected mice from death by MHV-A59 infection (FIG. 7B). The remaining mice were dissected 3 days after infection, livers were taken, weighed, ground to homogenate and centrifuged, and supernatants were taken to test for viral titer in tissues. The results show that doses of 0.01. mu.g/g and 0.05. mu.g/g are both effective in reducing the replication of MHV-A59 in mouse liver, and as the dose increases, the virus inhibition effect is better (FIG. 7C).

Reference:

1.Wang,Y.,et al.,Coronavirus nsp10/nsp16 Methyltransferase Can Be Targeted by nsp10-Derived Peptide In Vitro and In Vivo To Reduce Replication and Pathogenesis.J Virol,2015.89(16):p.8416-27.

2.Ge,X.Y.,et al.,Isolation and characterization of a bat SARS-like coronavirus that uses the ACE2 receptor.Nature,2013.503(7477):p.535-8.

3.Ommeh,S.,et al.,Genetic Evidence of Middle East Respiratory Syndrome Coronavirus(MERS-Cov)and Widespread Seroprevalence among Camels in Kenya.Virol Sin,2018.33(6):p.484-492.

4.Zhou,P.,et al.,Discovery of a novel coronavirus associated with the recent pneumonia outbreak in humans and its potential bat origin.bioRxiv,2020:p. 2020.01.22.914952.

sequence listing

<110> Wuhan university

Application of <120> Obatrox in preparation of broad-spectrum anti-coronavirus medicines

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

caatggttta acaggcacag g 21

<210> 2

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctcaagtgtc tgtggatcac g 21

Claims (4)

1. Application of olbatik mesylate shown in a structural formula (I) in preparing a medicament for preventing or treating coronavirus,

the coronavirus is human-infecting coronavirus selected from 2019 novel coronavirus 2019-nCoV, mouse hepatitis virus MHV, SARS-like coronavirus WIV1 from bat.

2. The application of a pharmaceutical composition in preparing a medicament for preventing or treating coronavirus is characterized in that the pharmaceutical composition contains olbuterol mesylate shown as a structural formula (I),

the coronavirus is human-infecting coronavirus selected from 2019 novel coronavirus 2019-nCoV, mouse hepatitis virus MHV, SARS-like coronavirus WIV1 from bat.

3. The use of claim 2, wherein said composition further comprises an additional antiviral agent.

4. The use of claim 3, wherein said composition further comprises a pharmaceutically acceptable carrier or excipient.

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